Expression system for factor VIII

ABSTRACT

This invention describes a protein-free production process for proteins having factor VIII procoagulant activity. The process includes the derivation of stable human cell clones with high productivity for B-domain deleted Factor VIII, and (2) the adaptation of cells to grow in a medium free of plasma-derived proteins.

RELATED APPLICATIONS

[0001] The application to Cho designated MSB-7241, “Human hybrid hostcell for mammalian gene expression,” and the application to Cho and Chandesignated MSB-7254, “Terminal repeat sequence of Epstein-Barr virusenhances drug selection ratio,” contain related subject matter. Bothapplications were filed on the same day as the current application.

BACKGROUND OF THE INVENTION

[0002] 1. Field

[0003] The present invention relates to an improved production methodfor factor VIII and its derivatives. The method relates generally tovector construction, transfection, and selection of cell lines withenhanced productivity under protein-free conditions. In particular, thisinvention relates to a process for preparing a protein with factor VIIIprocoagulant activity on an industrial scale.

[0004] 2. Background

[0005] Human factor VII is a trace plasma glycoprotein involved as acofactor in the activation of factor X and factor IXa. Inheriteddeficiency of factor VIII results in the X-linked bleeding disorderhemophilia A which can be treated successfully with purified factorVIII. The replacement therapy of hemophilia A has evolved from the useof plasma-derived factor VIII to the use of recombinant factor VIIIobtained by cloning and expressing the factor VIII cDNA in mammaliancells. (Wood et al., 1984, Nature 312: 330).

[0006] Factor VII has a domain organization of A1-A2-B-A3-C1-C2 and issynthesized as a single chain polypeptide of 2351 amino acids, fromwhich a 19-amino acid signal peptide is cleaved upon translocation intothe lumen of the endoplasmic reticulum. Due to the fact that factor VIIIis heaVIIIy glycosylated, high-level expression (>0.2 pg/c/d) of factorVIII has been difficult to achieve (Lind et al., 1995, Eur J Biochem.232: 19-27; Kaufman et al., 1989, Mol Cell Biol. 9: 1233-1242).Expression of factor VIII in mammalian cells is typically 2-3 orders ofmagnitude lower than that observed with other genes using similarvectors and approaches. The productivity of production cell lines forfactor VIII has been in the range of 0.5-1 μU/c/d (0.1-0.2 pg/c/d).

[0007] It has been demonstrated that the B-domain of factor VIII isdispensable for procoagulant activity. Using truncated variants offactor VIII, improved expression of factor VIII in mammalian cells hasbeen reported by various groups (Lind et al., 1995, Eur J Biochem 232:19-27; Tajima et al., 1990, Proc 6^(th) Int Symp H. T. p. 51-63; U.S.Pat. No. 5,661,008 to Almstedt, 1997). However, the expression level ofthe factor VIII variants remained below 1 pg/c/d from a stable cellclone.

SUMMARY OF THE INVENTION

[0008] We have now discovered (i) a method which derives cell lines withextremely high productivity of proteins having factor VIII procoagulantactivity, and (ii) a plasma protein-free production process for proteinshaving factor VIII procoagulant activity.

[0009] A process for the production of proteins having factor VIIIprocoagulant activity at the industrial scale. Using a newly createdcell host, cell clones with specific productivities in the range of 2-4pg/cell/day (10-20 μU/c/d) were derived. Under serum-free conditions,one clone has sustained a daily productivity of 2-4 pg/c/d. Clones withthis high level of productivity are able to produce 3-4 million unitsper day in a 15-liter perfusion fermenter. One unit of factor VIIIactivity is by definition the activity present in one milliliter ofplasma. One pg of factor VIII is generally equivalent to about 5 μU ofFVIII activity.

[0010] As used herein, a protein having factor VIII procoagulantactivity is a protein which causes the activation of Factor X in an invitro or in vivo model system. As non-limiting examples, this definitionincludes full length recombinant human factor VIII and the B domaindeleted factor VIII whose sequence is described in FIG. 1.

[0011] A high level of expression of a protein having factor VIIIprocoagulant activity means at least about 2 μU/c/d, or more preferablyat least about 4 μU/c/d, or most preferably at least about 5 μU/c/d, offactor VIII activity if grown in plasma derived protein-free medium, orat least about 4 μU/c/d, or more preferably at least about 8 μU/c/d, ormost preferably at least about 10 μU/c/d, of factor VIII activity ifgrown in medium supplemented with plasma derived protein. When theprotein expressed is BDD-FVIII, cell lines having specificproductivities up to about 15 μU/c/d, more preferably up to about 20μU/c/d may be obtained by the method described herein.

[0012] As used herein to describe the origin of cell lines, “derivedfrom” is intended to include, but not be limited to, normal mitotic celldivision and processes such as transfections, cell fusions, or othergenetic engineering techniques used to alter cells or produce cells withnew properties.

BRIEF DESCRIPTION OFTHE FIGURES

[0013]FIG. 1. Amino Acid Sequence of BDD-FVIII (SEQ ID NO:1).

[0014]FIG. 2. Sequence of terminal repeat (TR) sequence isolated fromEpstein-Barr virus (SEQ ID NO:2).

[0015]FIG. 3. Plasmid map of pCIS25DTR.

[0016]FIG. 4(a). Derivation of clone 20B8.

[0017]FIG. 4(b). Comparison of productivities of several clones invarious media. Three data points are presented from a two monthstability test of each clone.

[0018]FIG. 5. Volumetric productivity of clone 20B8.

SPECIFIC EMBODIMENTS

[0019] FVIII Assay

[0020] The activity of factor VIII derivatives obtained from recombinantgene expression in methotrexate (MTX)-resistant cell populations wasmeasured by a chromogenic assay. Activity was quantitated using Coatest®factor VIII:C/4 kit (Cromogenix, Molndal, Sweden) according tomanufacturer's instructions. A U.S. standard anti-hemophilic factor(factor VIII) known as MEGA 1 (Office of Biologics Research and Review,Bethesda, Md.) was used as the standard of measurement in this assay.See Barrowcliffe, 1993, Thromb Haem 70: 876.

[0021] Construction of Expression Vectors for B-domain Deleted FVIII

[0022] The sequence of the B-domain deleted (BDD) FVIII is shown inFIG. 1. The 90-kD and 80-kD chains were linked by a linker consisting of14 amino acids. See Chan, S. -Y., “Production of Recombinant Factor VIIIin the Presence of Liposome-like Substances of Mixed Composition,” U.S.patent application Ser. No. 08/634,001, filed Apr. 16, 1996. Theexpression vector for BDD-FVIII was made using standard recombinant DNAtechniques. The structure of the expression vector (pCIS25DTR) is shownin FIG. 3. The vector includes a transcriptional unit for BDD-FVIII anda selectable marker, dihydrofolate reductase (dhfr). In addition aterminal repeat sequence from Epstein-Barr virus, which shows enhanceddrug selection ratio, (FIG. 2) was inserted into the vector to increasethe integration efficiency. The vector is essentially a construct of avector (deposited ATCC 98879) which has been engineered to include atranscriptional unit corresponding to the sequence shown in FIG. 1.Further information about the terminal repeat sequence can be found inthe related patent application, incorporated herein by reference, to Choand Chan designated MSB-7254, “Terminal repeat sequence of Epstein-Barrvirus enhances drug selection ratio,” filed on the same day as thecurrent application.

[0023] Similar vectors can be constructed and used by those having skillin the art to obtain cells expressing proteins having factor VIIIprocoagulant activity. For example, coding sequences coding for knownvariants of factor VIII which retain procoagulant activity can besubstituted for the BDD-FVIII coding sequence. Also, instead of dhfr,other selectable markers can be used, such as glutamine synthetase (gs)or multidrug-resistance gene (mdr). The choice of a selection agent mustbe made accordingly, as is known in the art, i.e. for dhfr, thepreferred slection agent is methotrexate, for gs the preferred selectionagent is methionine sulfoximine, and for mdr the preferred selectionagent is colchicine.

WORKING EXAMPLES

[0024] Derivation of Cell Lines Expressing BDD-FVIII: Transfection, DrugSelection and Gene Amplification

[0025] Thirty micrograms of pCIS25DTR DNA was transferred into HKB11(ATCC deposit no. CRL 12568—a hybrid of 293S cells and human Burkitt'slymphoma cells, see U.S. patent application to Cho et al. filed on thesame day as the current application and designated MSB-7241,incorporated herein by reference) cells by electroporation set at 300volts and 300 micro farads (BTX Electro cell Manipulator 600) using a 2mm cuvette (BTX part #620). In comparative experiments done to parallelwork with the HKB11 cells, CHO (Chinese hamster ovary) and 293S (humanembryonic kidney) cells were transfected using a cationic lipid reagentDMRIE-C (Life Technologies, Gaithersburg, Md.) according to a protocolprovided by the Life Technologies. Amplification of transfected cellswas done with increasing methotrexate (MTX) concentrations (100 nM, 200nM, 400 nM, and 800 nM) at 1×10⁶ cells per 96 well plate in aMTX-selection medium lacking hypoxanthine and thymidine (DME/F12 mediawithout hypoxanthine and thymidine plus 5% dialyzed fetal bovine serumfrom Hyclone, Logan, Utah). MTX resistant cells were scored for growth,and secretion of the BDD-FVIII was screened using a Coatest® factor VIIIkit about 2-3 weeks post-transfection. The cultivation of cells weredone at 37° C. in a humidified 5% CO₂ incubator.

[0026] Limiting Dilution Cloning

[0027] Single cell clones (SCC) were derived by limiting dilutioncloning (LDC) of high producing populations in 96 well plates underserum-free conditions. Cells were seeded at 1-10 cells per well inDME/F12 media supplemented with Humulin® recombinant insulin (Lilly,Indianapolis, Ind.) at 10 μg/ml, 10× essential amino acids (LifeTechnology, Gaithersburg, Md.), and Plasmanate® human plasma proteinfraction (Bayer, Clayton, NC). Plasmanate® human plasma protein (HPP)fraction contains human albumin (88%) and various globulins (12%). Theclones were screened for BDD-FVIII productivity using the Coatest®factor VIII kits. The highest producing clones were selected forstability evaluation in shake flasks. For HKB cells, the first round LDCwas performed using selection medium supplemented with 5% dialyzed FBS.The second round LDC was done in serum-free but Plasmanate® HPPfraction-containing medium using the first SCC adapted in serum-freemedium supplemented with Plasmanate® HPP fraction.

[0028] Derivation of HKB clone 20B8

[0029] As summarized in FIG. 4(a), the initial population 1C10 wasderived from the HKB cells transfected with pCIS25DTR afteramplification with 400 nM MTX in the selection medium with 5% FBS. Oneof the first single cell clones (SCCs), 10A8, derived from 1C10 by a LDCusing a selection medium supplemented with 5% FBS was adapted inserum-free medium supplemented with Plasmanate® HPP fraction.Unexpectedly, 10A8 showed extremely increased levels of rFVIIIproduction at this stage (FIG. 4b). Therefore, we did a second LDC usingthe medium supplemented with Plasmanate® HPP fraction. The productivityof SCCs (e.g. 20B8) derived from the second LDC was similar withPlasmanate® HPP fraction-adapted 10A8. 20B8 showed higher levels ofBDD-FVIII than original 10A8 derived from the first LDC inserum-containing medium. Finally, 20B8 was adapted to growth in plasmaprotein-free (PPF) medium. Samples of 20B8 were deposited at theAmerican Type Culture Collection (Manassas, Va.) (ATCC deposit no.CRL-12582).

[0030] As shown in Table 1, HKB clones exhibit superior productivity forBDD-FVIII. A 10-20 fold increase in productivity was observed in HKBcells when compared to clones derived from transfected CHO and 293Scells. HKB cells, which do not form large aggregates of cells when grownin suspension culture, are preferred cells for the expression ofproteins having factor VIII procoagulant activity. TABLE 1 Expression ofFVIII and BDD-FVIII in human and rodent cell lines Specific Productivity(μU/c/d)* FVIII Derivatives BHK 293s CHO HKB Full length FVIII 0.45 1.20.5 1.0 BDD-FVIII ND 2.5 1.0 20

[0031] Plasma-protein-free Adaptation of Clones

[0032] HKB clones that have been adapted to grow as serum-freesuspension cultures were further weaned of plasma protein supplements.The weaning was done in sterile polycarbonate shake flasks (Corning,Corning, N.Y.) at a cell density of about 0.5×10⁶ cells/ml using plasmaderived protein free medium. The plasma protein free (PPF) medium wasDME/F12 medium supplemented with pluronic F68 (0.1%), CuSO₄ (50 nM), andFeSO₄/EDTA (50 μM). Complete medium exchange was done every 48 hours andthe shake flasks were re-seeded at 0.5×10⁶ cells/ml.

[0033] Fermentation of Clone 20B8

[0034] The productivity of clone 20B8 was evaluated in a 15-literperfusion fermenter. The fermenter was seeded with clone 20B8 cells at adensity of about 3×10⁶ cells/ml. The fermenter was perfused at a rate of4 volumes per day with the serum-free production medium as described inthe preceding paragraph. A final cell density of 2×10⁷ cells/ml wassustained throughout the evaluation period (45 days). As shown in FIG.5, during the first 4 weeks of fermentation, clone 20B8 was perfusedwith the serumfree production medium supplemented with Plasmanate® HPPfraction and was able to sustain high productivity. From day 28 to theend of the fermentation run, the cells were perfused with the sameserumfree production medium but without Plasmanate® HPP fraction. Asshown in FIG. 5, the cells continued to produce high levels of FVIII ina plasma derived protein-free environment. “Plasma derived protein-free”means that essentially no proteins isolated from plasma have been addedto the medium.

DISCUSSION

[0035] The derivation of HKB cells provides a protein-free productionsystem to produce not only BDD-FVIII but other therapeutic proteins aswell. Proteins produced from HKB cells have human glycosylation patternswhich may improve the half-life of certain glycoproteins in vivo. Thesecells should also be useful for the production of adenovirus andadeno-associated virus strains that have been designed for gene therapypurposes.

[0036] The above examples are intended to illustrate the invention andit is thought variations will occur to those skilled in the art.Accordingly, it is intended that the scope of the invention should belimited only by the claims below.

1 2 1 1438 PRT Artificial Sequence Description of Artificial SequenceDerived from human factor VIII sequence 1 Ala Thr Arg Arg Tyr Tyr LeuGly Ala Val Glu Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu GlyGlu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser PhePro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu PheThr Val His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro Trp Met GlyLeu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr Asp Thr Val ValIle Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 Ser Leu His Ala ValGly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 Glu Tyr Asp AspGln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 Phe Pro GlyGly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140 Gly ProMet Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170175 Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala AlaSer 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr ValAsn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys SerVal Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val HisSer Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His ArgGln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala GlnThr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe Cys His IleSer Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala Tyr Val Lys ValAsp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335 Met Lys Asn Asn GluGlu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Glu Met AspVal Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gln IleArg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr IleAla Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410415 Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420425 430 Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile435 440 445 Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu IleIle 450 455 460 Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro HisGly Ile 465 470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu ProLys Gly Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly GluIle Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro ThrLys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe ValAsn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro Leu LeuIle Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly Asn Gln IleMet Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser Val Phe Asp GluAsn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590 Arg Phe Leu ProAsn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605 Gln Ala SerAsn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 Leu GlnLeu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650655 Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660665 670 Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp675 680 685 Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met ThrAla 690 695 700 Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp TyrTyr Glu 705 710 715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu SerLys Asn Asn Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro ProVal Leu Lys Arg His 740 745 750 Gln Arg Glu Ile Thr Arg Thr Thr Leu GlnSer Asp Gln Glu Glu Ile 755 760 765 Asp Tyr Asp Asp Thr Ile Ser Val GluMet Lys Lys Glu Asp Phe Asp 770 775 780 Ile Tyr Asp Glu Asp Glu Asn GlnSer Pro Arg Ser Phe Gln Lys Lys 785 790 795 800 Thr Arg His Tyr Phe IleAla Ala Val Glu Arg Leu Trp Asp Tyr Gly 805 810 815 Met Ser Ser Ser ProHis Val Leu Arg Asn Arg Ala Gln Ser Gly Ser 820 825 830 Val Pro Gln PheLys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser 835 840 845 Phe Thr GlnPro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 850 855 860 Leu GlyPro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr 865 870 875 880Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile 885 890895 Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe 900905 910 Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His915 920 925 Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala TyrPhe 930 935 940 Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu IleGly Pro 945 950 955 960 Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro AlaHis Gly Arg Gln 965 970 975 Val Thr Val Gln Glu Phe Ala Leu Phe Phe ThrIle Phe Asp Glu Thr 980 985 990 Lys Ser Trp Tyr Phe Thr Glu Asn Met GluArg Asn Cys Arg Ala Pro 995 1000 1005 Cys Asn Ile Gln Met Glu Asp ProThr Phe Lys Glu Asn Tyr Arg Phe 1010 1015 1020 His Ala Ile Asn Gly TyrIle Met Asp Thr Leu Pro Gly Leu Val Met 1025 1030 1035 1040 Ala Gln AspGln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn 1045 1050 1055 GluAsn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg 1060 10651070 Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val1075 1080 1085 Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile TrpArg Val 1090 1095 1100 Glu Cys Leu Ile Gly Glu His Leu His Ala Gly MetSer Thr Leu Phe 1105 1110 1115 1120 Leu Val Tyr Ser Asn Lys Cys Gln ThrPro Leu Gly Met Ala Ser Gly 1125 1130 1135 His Ile Arg Asp Phe Gln IleThr Ala Ser Gly Gln Tyr Gly Gln Trp 1140 1145 1150 Ala Pro Lys Leu AlaArg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp 1155 1160 1165 Ser Thr LysGlu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1170 1175 1180 MetIle Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser 1185 11901195 1200 Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp GlyLys 1205 1210 1215 Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr LeuMet Val Phe 1220 1225 1230 Phe Gly Asn Val Asp Ser Ser Gly Ile Lys HisAsn Ile Phe Asn Pro 1235 1240 1245 Pro Ile Ile Ala Arg Tyr Ile Arg LeuHis Pro Thr His Tyr Ser Ile 1250 1255 1260 Arg Ser Thr Leu Arg Met GluLeu Met Gly Cys Asp Leu Asn Ser Cys 1265 1270 1275 1280 Ser Met Pro LeuGly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile 1285 1290 1295 Thr AlaSer Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser 1300 1305 1310Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln 13151320 1325 Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys ThrMet 1330 1335 1340 Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser LeuLeu Thr Ser 1345 1350 1355 1360 Met Tyr Val Lys Glu Phe Leu Ile Ser SerSer Gln Asp Gly His Gln 1365 1370 1375 Trp Thr Leu Phe Phe Gln Asn GlyLys Val Lys Val Phe Gln Gly Asn 1380 1385 1390 Gln Asp Ser Phe Thr ProVal Val Asn Ser Leu Asp Pro Pro Leu Leu 1395 1400 1405 Thr Arg Tyr LeuArg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1410 1415 1420 Leu ArgMet Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1425 1430 1435 2 402 DNAArtificial Sequence Description of Artificial Sequence Derived fromEpstein-Barr virus sequence 2 ggcaatggag cgtgacgaag ggccccagggctgaccccgg caaacgtgac ccggggctcc 60 ggggtgaccc aggcaagcgt ggccaaggggcccgtgggtg acacaggcaa ccctgacaaa 120 ggccccccag gaaagacccc cggggggcatcgggggggtg ttggcgggtc atgggggggg 180 cgggtcatgc cgcgcattcc tggaaaaagtggagggggcg tggccttccc cccgcggccc 240 cctagccccc ccgcagagag cggcgcaacggcgggcgagc ggcggggggt cggggtccgc 300 gggctccggg ggctgcgggc ggtggatggcggctggcgtt ccggggatcg ggggggggtc 360 ggggggcgct gcgcgggcgc agccatgcgtgaccgtgatg ag 402

We claim:
 1. A method of producing cells which express a protein havingfactor VIII procoagulant activity comprising the sequential steps of: a)obtaining cells which are solely of human origin, b) contacting thecells of step a) with a vector under conditions sufficient to allow thevector to enter the cells, wherein the vector comprises a selectablemarker and a sequence coding for the protein having factor VIIIprocoagulant activity operably linked to a promoter, c) selecting thecells from step b) with a selection agent, and d) isolating individualclones which express high levels of the protein having factor VIIIactivity from the cells obtained from step c).
 2. The method of claim 1,further comprising the step e) adapting the clones of step d) to growthin a plasma derived protein-free medium.
 3. The method of claim 1,wherein the cells of step a) are hybrids of human lymphoma cells and293S cells.
 4. The method of claim 1, wherein the cells of step a) arehybrids of 2B8 cells (ATCC CRL-12569) and 293S cells.
 5. The method ofclaim 1, wherein the cells of step a) are HKB11 cells (ATCC CRL-12568).6. The method of claim 1, wherein the steps c) are performed more thanonce.
 7. The method of claim 1, wherein the sequence of step b) codesfor the sequence shown in FIG. 1 (SEQ ID NO:1).
 8. The method of claim1, wherein the sequence of step b) codes for human factor VIII.
 9. Themethod of claim 1, wherein the selectable marker of step b) is dhfr andthe selection agent of step c) is methotrexate.
 10. The method of claim1, wherein the selectable marker of step b) is gs and the selectionagent of step c) is methionine sulfoximine.
 11. The method of claim 1,wherein the selectable marker of step b) is mdr and the selection agentof step c) is colchicine.
 12. A method of producing a protein havingfactor VIII activity comprising growing the cells produced by the methodof claim 1 in a growth medium and then isolating the protein havingfactor VIII activity from the medium.
 13. The method of claim 11 whereinthe protein is human factor VIII.
 14. The method of claim 11 wherein theprotein has the amino acid sequence shown in FIG. 1 (SEQ ID NO:1]). 15.A human cell line derived from human lymphoma cells and 293S cells whichexpresses high levels of a protein having factor VIII activity.
 16. Thehuman cell line of claim 14, wherein the human cell line is derived fromHKB11 cells (ATCC CRL-12568).
 17. A human cell line derived from humanlymphoma cells and 293S cells which expresses high levels of a proteinhaving factor VIII activity when grown in plasma derived protein-freemedium.
 18. The cell line of claim 16, wherein the cell line is derivedfrom HKB11 cells (ATCC CRL-12568).
 19. A cell line designated 20B8 (ATCCCRL-12582).